Indian Journal of Clinical Biochemistry, 2007 / 22 (1) Indian Journal of Clinical Biochemistry, 2007 / 22 (1) 22-28
CARDIOPROTECTIVE RESPONSE TO CHRONIC ADMINISTRATION OF VITAMIN E IN ISOPROTERENOL INDUCED MYOCARDIAL NECROSIS: HEMODYNAMIC, BIOCHEMICAL AND ULTRASTRUCTURAL STUDIES Mukesh Nandave, Ipseeta Mohanty, T. C. Nag1, Shreesh Kumar Ojha, Rajan Mittal, Santosh Kumari2, Dharamvir Singh Arya Department of Pharmacology and 1Anatomy, All India Institute of Medical Sciences, New Delhi-110 029 and 2 Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi-110 012 ABSTRACT The present study evaluated the cardioprotective potential of vitamin-E by studying its effect on hemodynamic parameters, lipid peroxidation, myocyte injury marker and ultrastructural changes in model of isoproterenolinduced myocardial necrosis in rats. Wistar albino male rats (150-200 g) were randomly divided into saline, ISP control, and vit E groups. Vitamin E group was administered vitamin E at a dose of 100 mg/kg/day while saline and ISP control groups received saline orally for one month. On 29th and 30th day, ISP (85 mg/kg, sc) was administered at an interval of 24 h to vit E and ISP control rats. On 31st day, rats of all groups were anesthetized and hemodynamic parameters were recorded. At the end of experimentation, animals were sacrificed; hearts were excised and processed for biochemical and ultrastructural studies. ISP administration produced marked cardiac necrosis as evidenced by significant decrease in myocardial creatine kinase- MB as well as increase in malonaldialdehyde levels. ISP-induced myocardial necrosis resulted in myocardial dysfunction as evidenced by significant depression in heart rate and mean arterial pressure in the ISP control group as compared to saline control. Salient ultrastructural changes including extensive loss of myofibrils, muscle necrosis, loss of mitochondria, and formation of several intracytoplasmic vacuoles and lipid droplets further confirmed the ISP-induced myocardial damage. However, subsequent to ISP challenge, vit E treatment significantly preserved the myocardium by restoring myocardial CK-MB activity, inhibiting the ISP-induced lipid peroxidation and ultrastructural changes. Additionally, pre-and co-treatment of vit E prevented the deleterious ultrastructural changes caused by ISP. These beneficial effects of chronic vit E treatment also translated into significant restoration of the altered hemodynamic parameters. The present study clearly demonstrated the cardioprotective potential of vit E at dose of 100 mg/kg in ISP-induced model of myocardial necrosis in rats. The significant restoration of altered hemodynamic parameters, myocardial CK-MB activity, prevention of ISP-induced rise in lipid peroxidation and ultrastructural changes may confirm its cardioprotective effect. KEY WORDS Vitamin E, Isoproterenol, Myocardial necrosis, Rats
INTRODUCTION Myocardial infarction (MI) is one of the most common manifestation of cardiovascular disease. The morbidity and
Address for Correspondence : Dr. D. S. Arya Associate Professor, Department of Pharmacology All India Institute of Medical Sciences, Ansari Nagar, New Delhi-110029, India E-mail :
[email protected] 22
mortality due to MI is now reaching epidemic proportion throughout the world. In recent years, accumulating evidence indicates that incidence and progression of cardiovascular disease may, to some extent, be modified by dietary means. In particular, attention has focused on the apparent beneficial effects of vitamin supplementation in reducing the incidence of cardiovascular disease (1). Vitamin E (vit E) is most widely used vitamin in food supplements and cosmetic products. Owing to its wide array of biological actions, public and scientific interest has been
Cardioprotective Response of Vitamin E
directed towards the role of vit E in health promotion and disease prevention. It is the predominant lipophilic antioxidant in plasma membrane and tissues and is the most abundant antioxidant in low-density lipoprotein (LDL). Besides having antioxidant properties, vit E has been shown to slow or inhibit the oxidative modification of LDL that is responsible for development and progression of atherosclerosis (2). Moreover, high levels of vit E have been measured in the mitochondria, golgi apparatus, lysosomes, and endoplasmic reticulum (3), and recent studies have shown that vit E possesses a variety of cardiovascular effects including decreased platelet aggregation (4), arterial superoxide generation and increased eNOS mediated NO production (5). Isoproterenol, a β-adrenergic agonist is a well-known inducer of myocardial necrosis and interstitial fibrosis at its supramaximal dosages. Mohanty et al. (6) have shown that isoproterenol leads to myocardial necrosis characterized by increased end-diastolic volume, end-diastolic pressure and left ventricular wall thickness. Some of the mechanisms proposed to explain the mechanisms of isoproterenol induced damage to cardiac myocytes include hypoxia due to myocardial hyperactivity and coronary hypotension, calcium overload, depletion of energy reserve and excessive production of free radicals resulting from oxidative metabolism of catecholamine. Free radical mediated peroxidation of membrane phospholipids and consequent changes in membrane permeability are the primary reasons for cardiotoxicity induced by isoproterenol (7-8). Oxidative stress also depresses the sarcolemmal Ca2+ transport and results in the development of intracellular Ca2+ overload and ventricular dysfunction (9). Oxidative stress has been implicated in the pathogenesis of myocardial ischemia. Therefore therapeutic interventions having antioxidant or free radical scavenging activity may exert beneficial effects against oxidative stress associated with various cardiovascular diseases, including ischemic heart disease (10-11).
other chemicals were of analytical grade. Animals : Wistar male albino rats, weighing 150 to 200g, 10 to 12 weeks old were used in the study. The study protocol was reviewed and approved by the Institutional Animal Ethics Committee and conforms with the Indian National Science Academy Guidelines for the Use and Care of Experimental Animals in research. Animals were obtained from the Central Animal House facility of All India Institute of Medical Sciences, New Delhi, India and were housed in polyacrylic cages (38x23x10 cm) with not more than four animals per cage. They were housed under standard laboratory conditions with natural light and dark cycles (approximately 12 h light/12 h dark) and maintained at humidity of 55+5% and an ambient temperature of 25+2oC. All experiments were performed between 9.00 and 16.00 h. The animals were allowed free excess to standard pellet diet (Ashirwad Industries Ltd.; India) and tap water ad libitum. The commercial pellet diet contained 24% protein, 5% fat, 4% fiber, 55% carbohydrate, 0.6% calcium, 0.3% phosphorous, 10% moisture and 9% ash w/w. The animals were allowed to acclimatize for one week before the experiments.
Based on these observations, vit E might have a protective effect on heart injury by ischemia. In the present study, isoproterenol-induced model of myocardial necrosis was used to investigate the cardio-protective effects of vit E. We also studied the effect of vit E treatment on hemodynamic parameters, lipid peroxidation, myocyte injury marker and ultrastructural changes to investigate mechanism of its cardioprotective effect.
Treatment protocol : A total of 36 animals were randomly allocated into three main groups comprising of twelve animals each. Animals of group 1 assigned as control were orally administered normal saline (0.9 % NaCl) once daily for a month. Animals of group 2 assigned as ISP control were orally administered normal saline once daily for a month and in addition received ISP (85 mg/kg, sc) on 29th and 30th day, at an interval of 24 h. Animals of group 3 assigned as drug treated group were orally administered vit E (100 mg/kg) suspended in normal saline with the help of carboxy methyl cellulose (CMC) once daily for 1 month and in addition challenged with ISP (85 mg/kg, sc) on 29th and 30th day, at an interval of 24 h. Hemodynamic parameters were recorded on the 31st day, i.e. 24 h after last injection of ISP. At the end of experimentation, animals were sacrificed under overdose of anesthesia; hearts were excised and immediately processed for biochemical and ultrastructural studies. In each group, out of twelve rats, 8 rats were used for hemodynamic and biochemical studies and remaining 4 rats for ultrastructural studies. The standardized isoproterenol dose 85 mg/kg subcutaneously as selected according to the previous studies (12-13). The selection of dose 100 mg/kg of vit E was based on the previous studies (6, 14)
MATERIALS AND METHODS Drug and chemicals : Vitamin E and isoproterenol (ISP) were procured from Sigma Chemicals (St Louis, MO, USA) and all
Hemodynamic studies : All animals were anesthetized intraperitoneally with pentobarbitone sodium (60 mg/kg). Atropine (4 mg/kg) was administered along with the anesthetic 23
Indian Journal of Clinical Biochemistry, 2007 / 22 (1)
to maintain heart rate especially during the surgery, and to reduce tracheo-bronchial secretions. The body temperature was monitored and maintained at 37 o C during the experimental protocol. The neck was opened with a ventral midline incision to perform tracheostomy and rats were ventilated with room air from a positive pressure ventilator (Inco, India) using compressed air at a rate of 70 strokes/min and a tidal volume of 10ml/kg. Ventilator setting and PO2 were adjusted as needed to maintain the arterial blood gas parameters within the physiological range. The left jugular vein was cannulated with polyethylene tube for continuous infusion of 0.9% saline solution. The right carotid artery was cannulated and the cannula was filled with heparinized saline and connected with CARDIOSYS CO-101 (Experimentria, Hungary) using a pressure transducer for the measurement of heart rate (HR), systolic arterial pressure (SAP), diastolic arterial pressure (DAP), and mean arterial pressure (MAP). Animals were allowed to stabilize for 15 minutes before recording the basal hemodynamic parameters. Biochemical studies : Hearts stored in liquid nitrogen were brought to room temperature and weighed. A 10% homogenate was prepared in ice-cold phosphate buffer (pH 7.4, 50 mM) and an aliquot of 0.2 ml was used for the assay of MDA. Estimation of lipid peroxidation marker Malonaldialdehyde (MDA) : Malonaldialdehyde (MDA) was estimated by the method of Ohkawa and colleagues (15). To different aliquot volume of the standard 1,1,3,3-tetramethoxypropane (1-10 nM), 0.2 ml of 8.1% sodium dodecyl sulphate, 1.5 ml of 20% acetic acid and 1.5 ml of 0.81% thiobarbituric acid were added. The mixture was heated for 30 min at 950 C in a temperature controlled water bath. After cooling, 5 ml of nbutanol : pyridine (15:1) was added to it. The mixture was then centrifuged at 5000 rpm for 10 min. Absorbance of the organic layer was read spectrophotometrically at excitation wavelength 532 nm and emission wavelength 515 nm. The readings of absorbance were plotted against concentration of tetramethoxypropane to produce standard curve. Estimation of myocyte injury marker: Creatine kinase-MB (CK-MB) : Creatine kinase-MB (CK-MB) was estimated spectrophotometrically using a standard enzyme kit supplied by Spinreact, S.A.-Spain (Cat.No.1001055), according to the method of Lamprecht and colleagues (16). The sample (50 μl) was added to cuvette containing 1.0 ml of prepared imidazole buffer consisting of 5.2 mM adenosine-monophosphate, 2.1 mM adenosine-di-phosphate, 2.1 mM nicotinamide adenine dinucleotide phosphate, 1.6 U/L glucose24
6-phosphate dehydrogenase, 31.2 mM creatine phosphate and 21 mM N-acetyl cysteine. It was incubated for 120 sec at room temperature and the absorbance was recorded at 340 nm for 180 sec at 60 sec intervals. One unit of CK-MB isoenzyme is defined, as the amount of enzyme at 30oC will transfer 1μmol of phosphate from phosphocreatine to ADP per min at pH 7.4. Ultrastructural studies (Transmission electron microscopy) : At the end of experiment, small pieces of myocardial tissue (approximately 1-2 mm in thickness) were immediately fixed in ice-cold Karnovsky’s fixative. The tissues were then washed in phosphate buffer (0.1M, pH 7.4) and postfixed for 2 h in 1% osmium tetroxide in the same buffer at 4°C. The specimens were then washed in phosphate buffer, dehydrated with graded acetone and then embedded in araldite CY 212 to make tissue blocks. The semithin as well as ultrathin sections (80-100nm) were cut by an ultramicrotome (Ultracut E, Reichert, Austria). The sections were stained with uranyl acetate and lead acetate and examined under transmission electron microscope (Morgagni 268D, Fei Co., The Netherlands) operated at 60KV. At least four hearts from each group were examined for ultrastructural changes. Statistical analysis : Descriptive statistics such as mean and standard deviation were calculated for all variables for each group. One-way Analysis of Variance (ANOVA) was applied for statistical analysis with post-hoc analysis (Bonferroni Multiple Range Test) for the evaluation of hemodynamic variables and Student’s t test was used for biochemical analysis. The p value
